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Volume 6, Number 1—February 2000
Letter

Carbapenem-Hydrolyzing Metallo-ß-Lactamase from a Nosocomial Isolate of Pseudomonas aeruginosa in France

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To the Editor: The carbapenems (meropenem and imipenem), the ß-lactams with the broadest spectrum, are stable to most ß-lactamases (1). Therefore, they are often used as antibiotics of last resort for treating nosocomial infections due to gram-negative bacteria resistant to other ß-lactams. Resistance to carbapenems and susceptibility to other ß-lactams in Pseudomonas aeruginosa is common as a result of reduced drug accumulation or increased expression of pump efflux (1).

Several extended-spectrum ß-lactamases have been reported in P. aeruginosa, but only two, IMP-1 and VIM-1, possess an extended hydrolysis profile that includes carbapenems (2-5). The chromosome-borne and plasmid-mediated carbapenem-hydrolyzing ß-lactamase, IMP-1, has been described in several gram-negative rods, including P. aeruginosa, P. cepacia, Alcaligenes xylosoxydans, and Enterobacteriaceae isolates in Japan (4,6). Recently, a chromosome-borne carbapenem-hydrolyzing ß-lactamase, VIM-1, was reported from a clinical isolate of P. aeruginosa in Italy (5), and uncharacterized carbapenem-hydrolyzing ß-lactamases have been reported in the United Kingdom and Portugal (7,8). The weakly related IMP-1 and VIM-1 (31.4% amino acid identity) are both zinc-dependent (metallo-enzymes) and confer resistance to all ß-lactams except monobactams (3,5).

In 1996, a 39-year-old French woman was hospitalized in Marseille for chronic myelogenous leukemia, pancytopenia, and allogeneic bone marrow transplantation. After a 15-day stay in the transplantation unit, fever developed and imipenem and amikacin were administered. Despite this treatment, the patient died of septic shock syndrome 5 days later. Three-day-old blood cultures grew a carbapenem-resistant P. aeruginosa isolate. This P. aeruginosa COL-1 isolate was resistant to most ß-lactams, including piperacillin/tazobactam, imipenem, meropenem, ceftazidime, cefepime (minimum inhibitory concentrations [MICs] of 128, 32, 16, 64, 32 mg/L, respectively), amikacin, tobramycin, gentamicin, netilmicin, and ciprofloxacin; however, the isolate was susceptible to aztreonam (MIC determination, genetic techniques and ß-lactamase assays are described elsewhere [9]). A sonicate of crude extract of P. aeruginosa COL-1 culture showed strong imipenem and meropenem hydrolysis activity (0.7 mU/mg and 1.9 mU/mg; reference P. aeruginosa strain <0.05 mU/mg) by UV spectrophotometry with 0.1 mM of substrate, after incubation in 50 mM phosphate buffer at 30°C. This activity was lost when the enzyme extract was preincubated with 10 mM of edetic acid and was partially restored by addition of 1 mM ZnCl2, indicating the presence of a metallo-carbapenem hydrolyzing ß-lactamase. Isoelectric focusing revealed two ß-lactamase bands of pI 5.6 and 9. Only the pI 5.6 ß-lactamase band was inhibited if the gel was overlaid with edetic acid before nitrocefin was added as the indicator substrate; the other pI 9 ß-lactamase likely corresponded to a naturally occurring AmpC cephalosporinase. This pI 5.6 value differed from the pI values of the carbapenem-hydrolyzing ß-lactamase previously reported in P. aeruginosa (3-5,7,8). Polymerase chain reaction amplification experiments were negative when internal primers were used for the only sequenced carbapenem-hydrolyzing ß-lactamase genes from P. aeruginosa encoding IMP-1 and VIM-1 and genomic DNA of P. aeruginosa COL-1. Transfer of the carbapenem resistance marker by conjugation to laboratory strains of P. aeruginosa or Escherichia coli was unsuccessful (9), but transformation by electroporation of a putative plasmid extract from P. aeruginosa COL-1 in E. coli, followed by selection onto amoxicillin-containing agar plates (9), gave a ca. 45-kb plasmid that produced the carbapenem-hydrolyzing ß-lactamase with a pI value of 5.6. Thus, the carbapenem-hydrolyzing ß-lactamase gene was plasmid-borne.

This case indicates the presence of a novel carbapenem-hydrolyzing ß-lactamase in P. aeruginosa in Europe, the first in France; its spread in gram-negative rods, as reported for IMP-1 in Japan, is of concern because, as seen in this case, routine laboratory detection is difficult and therapeutic options are extremely limited.

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Acknowledgment

This work was supported by a grant from the Ministère de l'Education Nationale, de la Recherche et de la Technologie, Université Paris XI, Faculté de Médecine Paris Sud, UPRES-JE-2227, France.

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Laurent Poirel*, Louis Collet†, and P. Nordmann*

Author affiliations: *Hôpital de Bicêtre, Le Kremlin-Bicêtre, France; †Institut Paoli-Calmettes, Marseille, France

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References

  1. Livermore  DM. Are all ß-lactams created equal? Scand J Infect Dis. 1996;Suppl 101S:3343.
  2. Nordmann  P, Guibert  M. Extended-spectrum ß-lactamases in Pseudomonas aeruginosa. J Antimicrob Chemother. 1998;42:12831. DOIPubMed
  3. Osano  E, Arakawa  Y, Wacharotayankun  R, Ohta  M, Horii  T, Ito  H, Molecular characterization of an enterobacterial metallo ß-lactamase found in a clinical isolate of Serratia marcescens that shows imipenem resistance. Antimicrob Agents Chemother. 1994;38:718.PubMed
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  5. Lauretti  L, Riccio  ML, Mazzariol  A, Cornaglia  G, Amicosante  G, Fontana  R, Cloning and characterization of blaVIM, a new integron-borne metallo-ß-lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrob Agents Chemother. 1999;43:158490.PubMed
  6. Senda  K, Arakawa  Y, Ichiyama  S, Nakashima  K, Ito  H, Shimokata  K, Characterization of metallo ß-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum ß-lactams. J Clin Microbiol. 1996;34:290913.PubMed
  7. Woodford  N, Palepou  MFI, Babini  GS, Bates  J, Livermore  DM. Carbapenemase-producing Pseudomonas aeruginosa in UK. Lancet. 1998;352:5467. DOIPubMed
  8. Cardoso  O, Sousa  JC, Leitao  R, Peixe  L. Carbapenem-hydrolysing ß-lactamase from clinical isolates of Pseudomonas aeruginosa in Portugal. J Antimicrob Chemother. 1999;44:135. DOIPubMed
  9. Philippon  LN, Naas  T, Bouthors  AT, Barakett  V, Nordmann  P. OXA-18, a class D clavulanic acid-inhibited extended-spectrum ß-lactamase from Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1997;41:218895.PubMed

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Cite This Article

DOI: 10.3201/eid0601.000117

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Table of Contents – Volume 6, Number 1—February 2000

Page created: May 09, 2011
Page updated: May 09, 2011
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